Team:Heidelberg/Templates/DelH week22

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Latest revision as of 21:36, 25 October 2013

23-09 - 29-09-13

Mutagenesis of DelH Clone I 6B

Screening-PCR Conditions CP.W22.A

Screening of the 11 colonies, which grew on the agar plate.

Result

Expected bands: 319 bp (HM22-VFII) and 395 bp (FS_66-SR_03)

Fig.22.1 Colony PCR for screening of DelH-pSB6A1 construct

10 ml of each colony were cultured.

• The screening with primer FS_66 to SR_03 did not work. The positive control was negative.
• The screening with primer VFII to HM22 was positive for 4 colonies (2,3,4,7,8,10)
• These colonies have to be midi-preped and digested to test whether it is the original DelH or the mutated one

Midiprep of Clones 2,3,4,7,8 and 10

• Centrifugation of 10 ml culures at 3750 rpm for 15 min
• Kept pellet an resuspended in 400 µl P1 buffer
• Added 800 µl P2 buffer and incubated for 180
• Neutralized with S3 buffer
• Centrifugated for 15 min at 13 000 rpm
• Kept supernatant and added 50:50 Isopropanol

Test restriction digest of Midipreps with MfeI and XbaI

Expected band: 22.9 kb

Fig.22.2 Test restriction digest with XbaI and MfeI to see if mutagenesis has worked.

• Incubation for 2 h at 37°C

Apparently the mutagenesis did not work. The band was too low. Maybe recombination occured.

Sequencing

The Midiprep of clone 2 was send to sequencing to see what had happened during the mutagenesis.

Sequencing results for colony 2 with primer VFII after mutagenesis.

Result

The mutagenesis did not work. Apparently recombination occured. As the result was the same for all the colonies we suspect that repeating the mutagenesis would lead to the same result. Therefore the mutagenesis part is stopped.

SDS-PAGE of Clone C5

Lysis of Cells

• 1 ml over night cultures in LB of electrocompetent BL21-pLys and BL21-pLys with pHM04 (C5)
• Meausured ODs (BL21-pLys = 1.33; BL21-pLys + pHM04= 1.03 )
• Centrifugation at 13000 rpm for 10 min
• Discarded supernatant
• Resuspended cells in 133 µl (BL21-pLys) and 103 µl (BL21-pLys + pHM04) 1x loading buffer (40% ddH₂O, 12.5% TrisHCl pH=7.5, 20% Glycerol (50%), 20% SDS (10%), 5% β-Mercaptoethanol, 2.5% Bromophenol blue)
• Boiled samples for 10 min at 98°C

Electrophoresis

• Applied 25 µl of each sample on SDS-polyacrylamide gel
• Run at 100 V for 5 min and afterwards at 140 V for another 50 min

Staining and Destaining Procedure

• Gel was stained in Coomassie Brilliant Blue for 1 h on shaker
• Destaining with destaining buffer (50% ddH₂O, 40% Methanol, 10% Acetic acid) until band were visible

Result

Fig.22.3: SDS page of DelH clone C5 in E. coli BL21 DE3

As shown in figure 22.3, there is a faint very high band, which is DelH (~600 kDa). Clone C5 expresses DelH despite of the amino acid substitution. Next, we will electroporate DelH C5 with DelRest and pIK8.6 in E. coli BL21 DE3 and analyze Delftibactin expression by Micro-TOF.

Generation of DelH Plasmid pHM04 18-09

Colony-PCR CP.W22.B

Result

Expected band: 663 bp

Fig.22.6 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E1-F12

Fig.22.5 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies C1-D12

Fig.22.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II(loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies A1-B12

• The following colonies seem positive, because they show the expected band at 663 bp

Test Restriction Digest

The positive screened colonies were digested with PvuI.

Result

Expected bands: 11,621, 8628 & 2,685 bp

Fig.22.7 Test restricion digest of colonies from plate II and III

The following colonies show the correct pattern:

They were not sequenced due to exclusively found mutations in BL21 DE3 (pLys).

Generation of DelH Plasmid pHM04 20-09

Colony-PCR CP.W22.C

Result

Expected band: 663 bp

Fig.22.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies G1-H12

Fig.22.3 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 4 µL of PCR)
l1:2-log ladder, l2-l26:colonies E1-F12

• The following colonies seem positive, because they show the expected band at 663 bp

Test Restriction Digest

The positive screened colonies were digested with PvuI.

Result

Expected bands: 11,621, 8628 & 2,685 bp

Fig.22.x Test restriction digest of DelH colonies

The following colonies show the correct pattern:

Sequencing

Colonies F7, F9, G3 and G4 were send for sequencing with VF2.

Result

Contamination of Cells with Chloramphenicol Resistance

Preparation of Electrocompetent BL21 DE3

Electrocompetent BL21 DE3 were prepared according to the protocol. The following day, the LB Chlor plate was covered with colonies. Since BL21 are in contrast to BL21 pLys not ntaurally resistant to Chloramphenicol, they are obviously contaminated. In order to check whether this is also caused by an Indigoidine plasmid, extensive evaluation of stocks and transformed cells will be undertaken.

Test for Chloramphenicol Resistance

All stocks of competent cells were streaked on LB Chlor plates and incubated at 37°C ON.

Result

Thus, the BL21 DE3 we obtained most probably are BL21 DE3 pLys, too.

New BL21 DE3

We obtained new BL21 DE3 from the Russel Lab, which were obtained from NEB directly. Cells were inocculated in 10 ml LB and streaked on LB Chlor plates. The vial was frozen again at -80°C.

Test for Indigoidine Contamination A

All competent cells as well as important transformed cells and recent mini preps of possible DelH plasmids were tested for contamination with an Indigoidine plasmid (which carries chloramphenicol resistance).

Result

Expected band: 540 bp

Fig.22.x Numerous samples were analyzed for contamination with Indigoidine.

The following samples show Indigoidine band:

All stocks are clean, only the DelH minipreps as well as the one from pIK2.6 are positive for Indigoidine. Therefore, we identified the source of contamination for the triple clone, which turned blue. We however cannot explain the positive result for the DelH minipreps, which obviously harbour a second plasmid.

Test for Indigoidine Contamination B

Again, all minipreps which were send for sequencing as well as a positive control a negative control and two recently prepared minipreps from other teams were tested for an Indigoidine contamination

Result

Expected band: 540 bp

Fig.22.x Test for contamination with Indigoidine plasmid

Surprisingly, all samples show Indigoidine band at ~540 bp. There is a contamination and we have to identify the source!

Test for Indigoidine Contamination C

Again, all minipreps which were send for sequencing as well as positive and negative controls and recently prepared minipreps from other teams were tested for an Indigoidine contamination. using two different primer pairs. Additionally, fake minipreps were performed to identify DNA contamination of buffers.

Result

Expected band: ~1 Kb for KH05 and 540 bp for RB69

Fig.22.x Testing for indigoidin Contamination uding RB69 and VF2

Fig.22.x Testing for indigoidin Contamination uding KH05 and VR

Amplification with KH05 shows contamination of new polymerase vial (shorter band) as well of DelH minipreps. Interestingly, the tested culture is NOT contaminated.